American Journal of Transplantation 2015; 15: 1349–1359 Wiley Periodicals Inc.
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Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13133
Anti-Phospholipase A2 Receptor Antibodies in Recurrent Membranous Nephropathy y
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A. Kattah1, , R. Ayalon2, , L. H. Beck Jr2, S. Sethi3, D. G. Sandor2, F. G. Cosio1, M. J. Gandhi4, E. C. Lorenz1, D. J. Salant2 and F. C. Fervenza1,* 1
Department of Nephrology and Hypertension, Mayo Clinic, Rochester, MN 2 Department of Medicine, Section of Nephrology, Boston University School of Medicine, Boston, MA 3 Department of Pathology, Mayo Clinic, Rochester, MN 4 Division of Transfusion Medicine, Rochester, MN Corresponding author: Fernando C. Fervenza, [email protected] y Both authors contributed equally. About 70% of patients with primary membranous nephropathy (MN) have circulating anti-phospholipase A2 receptor (PLA2R) antibodies that correlate with disease activity, but their predictive value in post-transplant (Tx) recurrent MN is uncertain. We evaluated 26 patients, 18 with recurrent MN and 8 without recurrence, with serial post-Tx serum samples and renal biopsies to determine if patients with pre-Tx anti-PLA2R are at increased risk of recurrence as compared to seronegative patients and to determine if post-Tx changes in anti-PLA2R correspond to the clinical course. In the recurrent group, 10/17 patients had anti-PLA2R at the time of Tx versus 2/7 patients in the nonrecurrent group. The positive predictive value of pre-Tx anti-PLA2R for recurrence was 83%, while the negative predictive value was 42%. Persistence or reappearance of post-Tx antiPLA2R was associated with increasing proteinuria and resistant disease in 6/18 cases; little or no proteinuria occurred in cases with pre-Tx antiPLA2R and biopsy evidence of recurrence in which the antibodies resolved with standard immunosuppression. Some cases with positive pre-Tx anti-PLA2R were seronegative at the time of recurrence. In conclusion, patients with positive pre-Tx anti-PLA2R should be monitored closely for recurrent MN. Persistence or reappearance of antibody post-Tx may indicate a more resistant disease. Abbreviations: CR, complete remission; ESRD, endstage renal disease; MN, membranous nephropathy; NPV, negative predictive value; NR, no response; PLA2R, M-type phospholipase A2 receptor; PPV, positive predictive value; PR, partial remission; RTX, rituximab; Tx, transplant
Received 28 July 2014, revised 06 November 2014 and accepted for publication 20 November 2014
Introduction Primary (idiopathic) membranous nephropathy (MN) is an organ-specific autoimmune disease caused by circulating autoantibodies that target glomerular podocyte antigens. The predominant target antigen is the M-type phospholipase A2 receptor (PLA2R) and anti-PLA2R antibodies are found in the serum of 70% patients with active primary MN (1–6). Moreover, PLA2R is typically detected in the glomerular immune deposits of patients with primary MN by immunohistology where it can serve as a footprint of PLA2R-associated MN even after patients have entered an immunological remission and serum anti-PLA2R is no longer detectable (1,7–9). About a third of patients with primary MN may develop end-stage renal disease (ESRD) (10,11) and many are suitable candidates for kidney transplantation. Unfortunately, recurrence can occur in approximately 40% of patients, usually within the first year (12). Recurrent MN varies in severity from a subclinical finding apparent only on protocol kidney biopsy to a severe disease manifesting by heavy proteinuria and a high risk of allograft failure (13,14). Case reports and small case series have shown that early identification of clinical recurrence and treatment with rituximab (RTX) can induce remission and avoid progressive kidney failure (14,15). However there has been no way to predict which patients with end-stage MN will develop recurrent MN or to detect its early occurrence other than by protocol biopsies. The primary purpose of this study was to determine if knowledge of a patient’s anti-PLA2R status at the time of transplantation has utility in forecasting their risk of recurrent MN. If, as suggested by previous case studies, anti-PLA2R antibodies are able to transfer the disease to the transplanted kidney (4,16,17), one would predict that those patients with persistently circulating antibodies at and after the time of transplantation would be at the greatest risk of recurrence. Given this theoretical risk, we set out to determine if MN recurs more frequently in patients that are seropositive for anti-PLA2R than in those that are
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seronegative at the time of transplantation. To do this we took advantage of a case series in which patients with endstage primary MN were followed with serial post-Tx protocol biopsies and serum collections before and after Tx. The longitudinal nature of the study enabled us to follow the clinical and serological outcome of the cases with histological and clinical recurrence.
Materials and Methods Patient population We identified 37 patients with ESRD who underwent kidney Tx at the Mayo Clinic due to primary MN between the years 2000 and 2010. We included all patients with available protocol biopsies, as well as at least one available stored serum sample in the Mayo Clinic Transplant Center Serum/Tissue Bank. Protocol biopsies were initiated at our institution in 1998 and occur at time of transplantation, 4, 12, 24 and 60 months post-Tx. We additionally attempted to retrieve native kidney biopsies from both Mayo Clinic and outside institutions. This study was conducted with the approval of the Mayo Clinic Institutional Review Board (IRB # 09-004518).
anti-human-IgG HRP conjugate that recognizes all human IgG subclasses. A highly positive index patient serum provided in the ELISA kit was used to generate a standard curve consisting of five calibrators (2, 20, 100, 500 and 1500 relative units [RU]/mL). In addition, we included our well-characterized positive control with each ELISA plate.
Tissue staining for PLA2R We examined all available serial sections from formalin fixed paraffin blocks (3 micron). Sections were deparaffinized with xylene and treated with proteinase K (20 mg/mL, 15 min at 378C; Invitrogen) for antigen retrieval. Sections were incubated with rabbit polyclonal anti-PLA2R1 (1:100; Sigma/ Atlas) for 30 min. As we anticipated weak PLA2R staining in patients with early stages of recurrence or in patients with resolving MN, amplification was used in all cases to optimize staining. The amplification process included sequential blocking with streptavidin and biotin (15 min; Streptavidin/Biotin blocking kit, Vector Laboratories) followed by biotinylated goat anti-rabbit IgG (1:200 for 45 min; Vector Laboratories) and DyLight 488 Streptavidin (1:100 for 45 min; Vector Laboratories).
Recurrent MN was defined by the presence of positive IgG staining in a capillary loop pattern by immunofluorescence (IF) and subepithelial deposits on electron microscopy (EM) (18).
All staining was done on coded sections without knowledge of the histological or clinical diagnosis and included a known positive control and known negative controls (lupus nephritis or normal human kidney). All glomeruli were photographed at 200 using an Olympus DP72 camera attached to a Nikon epifluorescence microscope. The coded images were scored as positive, negative, or equivocal by five observers, with high interobserver agreement. Any images on which there was disagreement were reviewed together by all five observers to reach a consensus.
Tx immunosuppression
HLA typing
The majority of patients with both recurrent and nonrecurrent MN received thymoglobulin for induction (14/18 vs. 6/8, respectively). Two patients received thymoglobulin and RTX for induction, one for a positive cross-match [4] and one for an ABO-incompatible Tx [24]. The remaining patients received either basiliximab [7, 10, 18] or daclizumab [25] for induction. The majority of patients (80%) received tacrolimus, mycophenolate mofetil and steroids for maintenance immunosuppression.
HLA typing of donors and recipients were reviewed and this data is provided in Table S1.
Biopsies
IgG subtyping We performed IgG subtyping (IgG subclass 1–4) on a single post-Tx biopsy specimen in patients with recurrent disease at the time point closest to the time of initial recurrence, when available. Results shown in Table S2.
Anti-PLA2R Western blotting and ELISA All available pre- and post-Tx serum samples were assayed both by Western blotting (WB) and with a commercial enzyme-linked immunosorbent assay (ELISA; Euroimmun US, Morris Plains, NJ). For WB, extracts of human glomeruli (containing native PLA2R) and recombinant human PLA2R (rPLA2R) were electrophoresed and immunoblotted with patients’ sera diluted at 1:25, as previously described(1). The anti-PLA2R level was semi-quantitated using a scale from 0 to 3 according to band intensity as compared to a well-characterized positive control. All weakly positive WB signals were repeated and confirmed as positive signals. In cases where all samples from an individual patient were found to be negative for the IgG4 subclass of anti-PLA2R, the pre-Tx sample was reassayed for total IgG anti-PLA2R to detect the potential presence of other IgG subclasses. In the patients with positive tissue staining for PLA2R and negative serum anti-PLA2R at the time of histologic recurrence with a previously positive serum anti-PLA2R (see below), the samples were repeated at dilution of 1:10 to increase sensitivity. ELISA for anti-PLA2R was carried out according to the manufacturer’s instructions. In brief, sera diluted to 1:100 were incubated 30 min with PLA2R coated microplates (Euroimmun US) and detected by incubation with
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Results Patient characteristics The data and samples for this study were from patients with primary MN that received a kidney Tx at the Mayo Clinic between 2000 and 2010. Of the 37 patients with idiopathic MN as their native renal disease, 21 had histologic recurrence post-Tx on protocol biopsy (57%) and 16 had no evidence of recurrence. Eleven patients were excluded due to lack of consent (n ¼ 10) or availability of serum (n ¼ 1), and therefore our analysis is based on a total of 26 patients: 18 with recurrent MN and 8 without recurrence (Table 1). The median time to recurrence, defined as a histologic recurrence regardless of level of proteinuria, was 4.1 months (IQR 2.6–38.5 months). The median proteinuria and mean serum creatinine at the time of recurrence was 533 mg/day (IQR 253–1679 mg/day) and 1.80 1.05 mg/ dL, respectively. American Journal of Transplantation 2015; 15: 1349–1359
Anti-PLA2R in Recurrent MN Table 1: Patient characteristics Characteristics Age at initial diagnosis Age at transplant Sex (no, % male) Race (no, % Caucasian) Donor type (no, %) Living related donor Living unrelated donor Cadaveric donor Biopsy-proven MN in native kidney (no, %) Prior transplants (no, %) Standard triple immunosuppressant therapy (no, %) Induction with Thymoglobulin (no, %)
Nonrecurrent n ¼ 8
Recurrent n ¼ 18
p-value*
41.5 11.3 52.5 10.5 6 (75%) 8 (100%)
41.7 13.6 51.3 11.1 11 (61%) 15 (83%)
0.87 0.80 0.67 0.53
3 (37.5%) 1 (12.5%) 4 (50%) 8 (100%) 0 6 (75%) 6 (75%)
10 (56%) 6 (33%) 2 (11%) 16 (89%) 4 (22%) 15 (83%) 14 (77%)
0.135
1.0 0.28 0.63 1.0
Mean standard deviation unless otherwise indicated. * Comparisons in baseline characteristics were performed by unpaired t-test, two tailed U test (Mann–Whitney) or Wilcoxon signed-rank test for continuous variables and Fisher’s exact test for binary variables.
Comparison of anti-PLA2R Western blotting and ELISA Using the manufacturer’s definition for anti-PLA2R negativity as 3 g/day and he was treated with a second course of RTX 3 years post-Tx, this time with achievement of a CR despite continued presence of the antibodies. Anti-PLA2R eventually resolved 3 years after the second RTX dose. His proteinuria worsened a third time to >3 g/day requiring a third RTX course. His protocol biopsies have shown features of ongoing MN on biopsy including positive PLA2R tissue staining (Figure 5). Positive pre-Tx anti-PLA2R without recurrent MN Two patients [20, 23] had strongly positive pre-Tx antiPLA2R but have not demonstrated evidence of recurrent MN on any protocol biopsies, starting at the 4-month point. However, in both cases, the first post-Tx serum sample was not available for anti-PLA2R testing until 2 and 5 years later, respectively, and both were seronegative. American Journal of Transplantation 2015; 15: 1349–1359
Negative pre-Tx anti-PLA2R with and without recurrent MN All seven patients with recurrent MN and five with no recurrence who were seronegative for anti-PLA2R pre-Tx remained seronegative during the post-Tx course.
Prior history of positive anti-PLA2R, negative pre-Tx with recurrent MN Patient 18 is unique in that this was his second Tx, as his first was lost due to venous thrombosis. Prior to his first Tx, he was anti-PLA2R seropositive, but during the short period between transplants, the antibody disappeared such that he was seronegative at the time of his second Tx. His 4-month protocol biopsy showed recurrent MN with positive PLA2R tissue staining, although his serum remained negative for anti-PLA2R and he has not developed significant proteinuria.
Discussion The primary outcome of this study to determine the predictive value of pre-Tx anti-PLA2R in recurrent MN is that recurrence occurs in a high proportion of patients with positive pre-Tx antibody (83%), but also that the risk of recurrence is as high as 58% in those that are seronegative pre-Tx (Figure 1). In our cohort, the PPV of positive pre-Tx anti-PLA2R for recurrent MN is 83% and the NPV is 42%. Though the number of patients is small, further analysis of follow up data allows us to make 1355
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Figure 4: Patient 5. Recurrence diagnosed at 0.3 months post-Tx. (A) Changes in proteinuria, semi-quantitative Ab levels and IgG staining by immunofluorescence (IF) over time. (B) Weakly-positive PLA2R staining at 0.3 months post-Tx in setting of antibodymediated rejection. (C) Positive PLA2R staining at 18 months postTx.
broader statements that will be of value in the future. Although a positive test for anti-PLA2R pre-Tx predicts early recurrence of MN, in some cases this is a subclinical biopsy finding that may resolve with standard post-Tx immunosuppression. It has been previously established that symptomatic recurrence may lead to graft loss (12,13,21–23). In this study graft loss was an infrequent outcome (3/18 patients) because most cases of recurrence that were detected by protocol biopsy were effectively treated with RTX if they developed proteinuria (11/18 patients). A particularly informative subset of patients was those that were seropositive pre-Tx and became seronegative without additional immunosuppression [14, 18, 20, 23]. In these cases, prospective serial measurements showing decline and disappearance of anti-PLA2R could be used clinically in place of biopsies to monitor for recurrent disease. We propose that patients with primary MN may be separated into two distinct groups—those with PLA2Rassociated MN and those with non-PLA2R-associated MN. We define those with PLA2R-associated MN in this 1356
Figure 5: Patient 8. Recurrence diagnosed at 3 months post-Tx. (A) Changes in proteinuria, semi-quantitative Ab levels and IgG staining by immunofluorescence (IF) over time. (B) Positive PLA2R staining at 16 months post-Tx, after RTX. (C) Positive PLA2R staining 5 years post-Tx, after two courses of RTX.
study as patients with a positive serum anti-PLA2R either pre- or post-Tx and/or positive staining for PLA2R in kidney tissue in the native biopsy or at the time of recurrence. Patients with documented native or recurrent MN on biopsy with consistently negative serum and negative tissue staining are considered to have non-PLA2Rassociated MN. Those patients who were seronegative pre-Tx and had no evidence of recurrence or no tissue available for staining at time of recurrence could potentially fall into either group, only prior information on anti-PLA2R or PLA2R staining of the native kidney biopsy at the time of original diagnosis could have distinguished them. Due to the lack of serum and limited availability of tissue samples from the time when patients were initially diagnosed with MN in their native kidneys, the recurrence rate amongst those with PLA2R-associated and non-PLA2R associated MN cannot be determined in this cohort, although there was close concordance between the native kidney PLA2R positivity and pretransplant anti-PLA2R status in the few cases in which tissue American Journal of Transplantation 2015; 15: 1349–1359
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was available. However, we were able to identify a single subject [22] whose native biopsy stained for PLA2R thus establishing the disease as PLA2R-associated MN. The finding of seronegativity at transplantation suggests a lack of immunologic activity, and is likely the reason why this patient had no evidence of recurrence. Our study includes 18 patients with recurrence: 12 of them with PLA2R-associated MN (66%), four with non-PLA2R associated MN (22%), and two with unknown status (11%). This rate is compatible with the prevalence of antiPLA2R in patients with primary MN. The overall recurrence rate in our cohort (57%) is higher than in previously published series (30–42%) (13,24), but could be related to earlier histologic diagnosis with protocol biopsies, longer follow up of our cohort, and the high number of living related donor transplants in this cohort (25). Our biopsy protocol also provided a unique opportunity to verify whether the IgG1 subclass is dominant and predates IgG4 deposition in glomeruli as suggested by Huang et al in native biopsies (20). Of the seven patients (regardless of PLA2R status) that had biopsies available for IgG subtyping at the first time histologic recurrence was noted [5, 6, 11, 14–17], 6/7 had predominantly IgG4 staining in capillary walls (Table S2). Similarly, IgG4 was the only or dominant subclass in most of the earliest biopsies of subjects with PLA2R-associated MN [5, 6, 9, 11, 14, 15]. A single case of known non-PLA2R-associated MN [16] had only IgG1 staining. Recurrent MN In patients with positive pre-Tx anti-PLA2R, the antibody can disappear within 13 months or less after Tx, which may be due to standard post-Tx immunosuppression, additional RTX treatment, or unknown factors related to antigen exposure in the allograft. Early recurrence that resolved spontaneously could have been missed in some cases prior to the first protocol biopsy. In addition, a few patients were seronegative at time of recurrence and had positive tissue staining for PLA2R [11, 13, 14, 18]. In two of them [11, 14] one might argue that their high pre-Tx antibody levels induced an early recurrence but were no longer detectable in the serum by the time of the first protocol biopsy. Such reasoning cannot apply to patient 13 who recurred after 26 months and whose 4-month protocol biopsy did not have evidence of MN. Nor does it explain why patient 18 developed PLA2R-associated recurrent MN in his second allograft at a time when he was apparently seronegative and thus his kidney was never exposed to detectable antiPLA2R. This finding of negative anti-PLA2R in the serum with positive tissue staining is consistent with prior studies (4) and may reflect very low levels of circulating antibodies that are consumed by the allograft and thus are undetectable by the most sensitive methods available. These low levels may be able to accumulate in the American Journal of Transplantation 2015; 15: 1349–1359
glomeruli over time and not only cause histological changes detectable by immunostaining but can also induce clinical recurrence and graft loss despite apparent seronegativity. These data also suggest that tissue staining for PLA2R may be a more sensitive modality for detecting early recurrence, though this method may be less useful in centers that do not perform protocol biopsies. The persistence or reappearance of anti-PLA2R may herald a more aggressive disease. In six patients [5, 7–10, 15], antiPLA2R levels remained positive or re-appeared years after Tx and were accompanied by symptomatic disease with significant proteinuria. Another insight from this study is that about half of those patients that were seronegative pre-Tx and post-Tx went on to develop symptomatic recurrent MN. As up to 20% of patients with idiopathic MN may have non-PLA2R-associated disease, we would expect that some of the patients in our cohort also have MN associated with one or more distinct, and as yet unidentified, podocyte antigens. However, assuming that at least some of these seronegative cases did indeed have PLA2R-associated MN in immunologic remission at the time of transplantation, this study provided an opportunity to determine if they reactivated when exposed to the new allograft. Notably, none of these cases became seropositive post-Tx, and none of those with available tissue had positive PLA2R immunostaining. These results suggest that post-Tx antiPLA2R testing is of little value in cases that are seronegative pre-Tx unless they are known (from prior serological studies or PLA2R staining of their native kidney) to have PLA2Rassociated MN. Nonrecurrent MN There were two patients that were seropositive for antiPLA2R pre-Tx that became seronegative post-Tx and had no evidence of recurrent MN on protocol biopsy, which suggests that the antibodies either failed to transfer the disease to the allograft or Tx immunosuppression rapidly induced suppression of anti-PLA2R production. In those patients seronegative for anti-PLA2R at the time of Tx, a proportion were likely cases of PLA2R-associated who were immunologically inactive, such as patient 22 who was positive for the PLA2R antigen on native biopsy but seronegative at the time of Tx. While in anti-glomerular basement membrane (GBM) disease, the recurrence rate is significantly reduced if circulating anti-GBM antibody levels have been undetectable for at least 12 months before Tx (26), the optimal duration of seronegativity in PLA2Rassociated MN is unknown. Our findings are consistent with the findings of Debiec et al, who have previously shown that the kinetics of anti-PLA2R post-Tx is quite variable and we found similar patterns of change anti-PLA2R post-Tx (4). Our study has the advantage of using protocol biopsies, allowing us to identify 1357
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subclinical, histologic recurrence of MN, as well as more complete serologic follow up of anti-PLA2R, including both pre- and post-Tx samples. Our study has several limitations. It is a small, retrospective study and so our analysis is limited by the availability of serum and tissue samples. The data is quite heterogeneous and our power to detect significant differences between groups is limited. In addition, we are unable to comment on the overall recurrence rate in PLA2R-associated versus nonPLA2R-associated MN due to limited native kidney biopsy specimens. In conclusion, pre-Tx anti-PLA2R testing may have clinical utility either in those with known PLA2R-associated disease or those who are seropositive at the time of transplant. Now that the assays are more widely available in the clinical setting, anti-PLA2R should be checked in all patients with primary MN being considered for transplantation and the native kidney biopsy should be stained for PLA2R in order to classify them as having PLA2R- or nonPLA2R-associated MN prior to transplantation. The presence of anti-PLA2R at the time of Tx very often leads to early recurrence of MN. Induction therapy and standard maintenance immunosuppression in some cases are sufficient to induce immunologic remission, but do not preclude the possibility of recurrence of PLA2R-associated MN. In those cases with persistence or reappearance of the antibody post-Tx the disease appears more resistant. The response to RTX is excellent and should be considered to prevent allograft loss. Our study raises two practical issues. One is whether to postpone Tx until the circulating anti-PLA2R disappears and for how long to wait with an undetectable level before Tx? Second, considering the high recurrence rate documented in patients seropositive for anti-PLA2R at the time of transplantation, should RTX be given preventively to all patients with either pre- or post-Tx anti-PLA2R seropositivity? Future prospective studies are needed to establish the answers to these questions.
Acknowledgments We would like to thank Dr. Joel Henderson from the Department of Pathology and Laboratory Medicine at Boston University Medical Center for guidance and advice with the tissue staining. This work is supported by R01 DK090029 (DJS, LHB), R01 DK 097053 (LHB) from NIH/NIDDK and NEPTUNE, Nephrotic Syndrome Study Network, U-54-DK-083912 (RA).
Disclosure The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. LHB and DJS are co-inventors on the US patent, Diagnostics for Membranous Nephropathy. 1358
References 1. Beck LH Jr, Bonegio RG, Lambeau G, Beck DM, Powell DW, Cummins TD. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med 2009; 361: 11–21. 2. Hofstra JM, Beck LH Jr, Beck DM, Wetzels JF, Salant DJ. Antiphospholipase A(2) receptor antibodies correlate with clinical status in idiopathic membranous nephropathy. Clin J Am Soc Nephrol 2011; 6: 1286–1291. 3. Hoxha E, Harendza S, Zahner G, Panzer U, Steinmetz O, Fechner K. An immunofluorescence test for phospholipase-A(2)-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis. Nephrol Dial Transplant 2011; 26: 2526–2532. 4. Debiec H, Martin L, Jouanneau C, et al. Autoantibodies specific for the phospholipase A2 receptor in recurrent and De Novo membranous nephropathy. Am J Transplant 2011; 11: 2144–2152. 5. Qin W, Beck LH Jr, Zeng C, et al. Anti-phospholipase A2 receptor antibody in membranous nephropathy. J Am Soc Nephrol 2011; 22: 1137–1143. 6. Oh YJ, Yang SH, Kim DK, Kang SW, Kim YS. Autoantibodies against phospholipase A2 receptor in Korean patients with membranous nephropathy. PLoS ONE 2013; 8: e62151. 7. Svobodova B, Honsova E, Ronco P, Tesar V, Debiec H. Kidney biopsy is a sensitive tool for retrospective diagnosis of PLA2Rrelated membranous nephropathy. Nephrol Dial Transplant 2013; 28: 1839–1844. 8. Debiec H, Ronco P. PLA2R autoantibodies and PLA2R glomerular deposits in membranous nephropathy. N Engl J Med 2011; 364: 689–690. 9. Larsen CP, Messias NC, Silva FG, Messias E, Walker PD. Determination of primary versus secondary membranous glomerulopathy utilizing phospholipase A2 receptor staining in renal biopsies. Mod Pathol 2013; 26: 709–715. 10. Donadio JV Jr, Torres VE, Velosa JA, et al. Idiopathic membranous nephropathy: the natural history of untreated patients. Kidney Int 1988; 33: 708–715. 11. Schieppati A, Mosconi L, Perna A, et al. Prognosis of untreated patients with idiopathic membranous nephropathy. N Engl J Med 1993; 329: 85–89. 12. Dabade TS, Grande JP, Norby SM, Fervenza FC, Cosio FG. Recurrent idiopathic membranous nephropathy after kidney transplantation: a surveillance biopsy study. Am J Transplant [Research Support. Non-U.S. Gov’t]. 2008; 8: 1318–1322. 13. Poduval RD, Josephson MA, Javaid B. Treatment of de novo and recurrent membranous nephropathy in renal transplant patients. Semin Nephrol 2003; 23: 392–399. 14. El-Zoghby ZM, Grande JP, Fraile MG, Norby SM, Fervenza FC, Cosio FG. Recurrent idiopathic membranous nephropathy: Early diagnosis by protocol biopsies and treatment with antiCD20 monoclonal antibodies. Am J Transplant 2009; 9: 2800–2807. 15. Sprangers B, Lefkowitz GI, Cohen SD, et al. Beneficial effect of rituximab in the treatment of recurrent idiopathic membranous nephropathy after kidney transplantation. Clin J Am Soc Nephrol 2010; 5:790–797. 16. Stahl R, Hoxha E, Fechner K. PLA2R autoantibodies and recurrent membranous nephropathy after transplantation. N Engl J Med 2010; 363: 496–498. 17. Seitz-Polski B, Payre C, Ambrosetti D, et al. Prediction of membranous nephropathy recurrence after transplantation by monitoring of anti-PLA2R1 (M-type phospholipase A2 receptor)
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Supporting Information Additional Supporting Information may be found in the online version of this article. Supplementary Material and Methods Figure S1: Correlation between Western blot (semiquantitative scale) and ELISA. ELISA values were logarithmically transformed due to non-normal distribution. Table S1: HLA typing in recipients and donors. Table S2: PLA2R antigen staining on native biopsies and IgG subtyping in posttransplant biopsies in patients with MN recurrence. Table S3: Anti-PLA2R results by Western blotting (semiquantitative compared to well-characterized positive control) and by ELISA (RU/mL; negative
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Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13133
Anti-Phospholipase A2 Receptor Antibodies in Recurrent Membranous Nephropathy y
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A. Kattah1, , R. Ayalon2, , L. H. Beck Jr2, S. Sethi3, D. G. Sandor2, F. G. Cosio1, M. J. Gandhi4, E. C. Lorenz1, D. J. Salant2 and F. C. Fervenza1,* 1
Department of Nephrology and Hypertension, Mayo Clinic, Rochester, MN 2 Department of Medicine, Section of Nephrology, Boston University School of Medicine, Boston, MA 3 Department of Pathology, Mayo Clinic, Rochester, MN 4 Division of Transfusion Medicine, Rochester, MN Corresponding author: Fernando C. Fervenza, [email protected] y Both authors contributed equally. About 70% of patients with primary membranous nephropathy (MN) have circulating anti-phospholipase A2 receptor (PLA2R) antibodies that correlate with disease activity, but their predictive value in post-transplant (Tx) recurrent MN is uncertain. We evaluated 26 patients, 18 with recurrent MN and 8 without recurrence, with serial post-Tx serum samples and renal biopsies to determine if patients with pre-Tx anti-PLA2R are at increased risk of recurrence as compared to seronegative patients and to determine if post-Tx changes in anti-PLA2R correspond to the clinical course. In the recurrent group, 10/17 patients had anti-PLA2R at the time of Tx versus 2/7 patients in the nonrecurrent group. The positive predictive value of pre-Tx anti-PLA2R for recurrence was 83%, while the negative predictive value was 42%. Persistence or reappearance of post-Tx antiPLA2R was associated with increasing proteinuria and resistant disease in 6/18 cases; little or no proteinuria occurred in cases with pre-Tx antiPLA2R and biopsy evidence of recurrence in which the antibodies resolved with standard immunosuppression. Some cases with positive pre-Tx anti-PLA2R were seronegative at the time of recurrence. In conclusion, patients with positive pre-Tx anti-PLA2R should be monitored closely for recurrent MN. Persistence or reappearance of antibody post-Tx may indicate a more resistant disease. Abbreviations: CR, complete remission; ESRD, endstage renal disease; MN, membranous nephropathy; NPV, negative predictive value; NR, no response; PLA2R, M-type phospholipase A2 receptor; PPV, positive predictive value; PR, partial remission; RTX, rituximab; Tx, transplant
Received 28 July 2014, revised 06 November 2014 and accepted for publication 20 November 2014
Introduction Primary (idiopathic) membranous nephropathy (MN) is an organ-specific autoimmune disease caused by circulating autoantibodies that target glomerular podocyte antigens. The predominant target antigen is the M-type phospholipase A2 receptor (PLA2R) and anti-PLA2R antibodies are found in the serum of 70% patients with active primary MN (1–6). Moreover, PLA2R is typically detected in the glomerular immune deposits of patients with primary MN by immunohistology where it can serve as a footprint of PLA2R-associated MN even after patients have entered an immunological remission and serum anti-PLA2R is no longer detectable (1,7–9). About a third of patients with primary MN may develop end-stage renal disease (ESRD) (10,11) and many are suitable candidates for kidney transplantation. Unfortunately, recurrence can occur in approximately 40% of patients, usually within the first year (12). Recurrent MN varies in severity from a subclinical finding apparent only on protocol kidney biopsy to a severe disease manifesting by heavy proteinuria and a high risk of allograft failure (13,14). Case reports and small case series have shown that early identification of clinical recurrence and treatment with rituximab (RTX) can induce remission and avoid progressive kidney failure (14,15). However there has been no way to predict which patients with end-stage MN will develop recurrent MN or to detect its early occurrence other than by protocol biopsies. The primary purpose of this study was to determine if knowledge of a patient’s anti-PLA2R status at the time of transplantation has utility in forecasting their risk of recurrent MN. If, as suggested by previous case studies, anti-PLA2R antibodies are able to transfer the disease to the transplanted kidney (4,16,17), one would predict that those patients with persistently circulating antibodies at and after the time of transplantation would be at the greatest risk of recurrence. Given this theoretical risk, we set out to determine if MN recurs more frequently in patients that are seropositive for anti-PLA2R than in those that are
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seronegative at the time of transplantation. To do this we took advantage of a case series in which patients with endstage primary MN were followed with serial post-Tx protocol biopsies and serum collections before and after Tx. The longitudinal nature of the study enabled us to follow the clinical and serological outcome of the cases with histological and clinical recurrence.
Materials and Methods Patient population We identified 37 patients with ESRD who underwent kidney Tx at the Mayo Clinic due to primary MN between the years 2000 and 2010. We included all patients with available protocol biopsies, as well as at least one available stored serum sample in the Mayo Clinic Transplant Center Serum/Tissue Bank. Protocol biopsies were initiated at our institution in 1998 and occur at time of transplantation, 4, 12, 24 and 60 months post-Tx. We additionally attempted to retrieve native kidney biopsies from both Mayo Clinic and outside institutions. This study was conducted with the approval of the Mayo Clinic Institutional Review Board (IRB # 09-004518).
anti-human-IgG HRP conjugate that recognizes all human IgG subclasses. A highly positive index patient serum provided in the ELISA kit was used to generate a standard curve consisting of five calibrators (2, 20, 100, 500 and 1500 relative units [RU]/mL). In addition, we included our well-characterized positive control with each ELISA plate.
Tissue staining for PLA2R We examined all available serial sections from formalin fixed paraffin blocks (3 micron). Sections were deparaffinized with xylene and treated with proteinase K (20 mg/mL, 15 min at 378C; Invitrogen) for antigen retrieval. Sections were incubated with rabbit polyclonal anti-PLA2R1 (1:100; Sigma/ Atlas) for 30 min. As we anticipated weak PLA2R staining in patients with early stages of recurrence or in patients with resolving MN, amplification was used in all cases to optimize staining. The amplification process included sequential blocking with streptavidin and biotin (15 min; Streptavidin/Biotin blocking kit, Vector Laboratories) followed by biotinylated goat anti-rabbit IgG (1:200 for 45 min; Vector Laboratories) and DyLight 488 Streptavidin (1:100 for 45 min; Vector Laboratories).
Recurrent MN was defined by the presence of positive IgG staining in a capillary loop pattern by immunofluorescence (IF) and subepithelial deposits on electron microscopy (EM) (18).
All staining was done on coded sections without knowledge of the histological or clinical diagnosis and included a known positive control and known negative controls (lupus nephritis or normal human kidney). All glomeruli were photographed at 200 using an Olympus DP72 camera attached to a Nikon epifluorescence microscope. The coded images were scored as positive, negative, or equivocal by five observers, with high interobserver agreement. Any images on which there was disagreement were reviewed together by all five observers to reach a consensus.
Tx immunosuppression
HLA typing
The majority of patients with both recurrent and nonrecurrent MN received thymoglobulin for induction (14/18 vs. 6/8, respectively). Two patients received thymoglobulin and RTX for induction, one for a positive cross-match [4] and one for an ABO-incompatible Tx [24]. The remaining patients received either basiliximab [7, 10, 18] or daclizumab [25] for induction. The majority of patients (80%) received tacrolimus, mycophenolate mofetil and steroids for maintenance immunosuppression.
HLA typing of donors and recipients were reviewed and this data is provided in Table S1.
Biopsies
IgG subtyping We performed IgG subtyping (IgG subclass 1–4) on a single post-Tx biopsy specimen in patients with recurrent disease at the time point closest to the time of initial recurrence, when available. Results shown in Table S2.
Anti-PLA2R Western blotting and ELISA All available pre- and post-Tx serum samples were assayed both by Western blotting (WB) and with a commercial enzyme-linked immunosorbent assay (ELISA; Euroimmun US, Morris Plains, NJ). For WB, extracts of human glomeruli (containing native PLA2R) and recombinant human PLA2R (rPLA2R) were electrophoresed and immunoblotted with patients’ sera diluted at 1:25, as previously described(1). The anti-PLA2R level was semi-quantitated using a scale from 0 to 3 according to band intensity as compared to a well-characterized positive control. All weakly positive WB signals were repeated and confirmed as positive signals. In cases where all samples from an individual patient were found to be negative for the IgG4 subclass of anti-PLA2R, the pre-Tx sample was reassayed for total IgG anti-PLA2R to detect the potential presence of other IgG subclasses. In the patients with positive tissue staining for PLA2R and negative serum anti-PLA2R at the time of histologic recurrence with a previously positive serum anti-PLA2R (see below), the samples were repeated at dilution of 1:10 to increase sensitivity. ELISA for anti-PLA2R was carried out according to the manufacturer’s instructions. In brief, sera diluted to 1:100 were incubated 30 min with PLA2R coated microplates (Euroimmun US) and detected by incubation with
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Results Patient characteristics The data and samples for this study were from patients with primary MN that received a kidney Tx at the Mayo Clinic between 2000 and 2010. Of the 37 patients with idiopathic MN as their native renal disease, 21 had histologic recurrence post-Tx on protocol biopsy (57%) and 16 had no evidence of recurrence. Eleven patients were excluded due to lack of consent (n ¼ 10) or availability of serum (n ¼ 1), and therefore our analysis is based on a total of 26 patients: 18 with recurrent MN and 8 without recurrence (Table 1). The median time to recurrence, defined as a histologic recurrence regardless of level of proteinuria, was 4.1 months (IQR 2.6–38.5 months). The median proteinuria and mean serum creatinine at the time of recurrence was 533 mg/day (IQR 253–1679 mg/day) and 1.80 1.05 mg/ dL, respectively. American Journal of Transplantation 2015; 15: 1349–1359
Anti-PLA2R in Recurrent MN Table 1: Patient characteristics Characteristics Age at initial diagnosis Age at transplant Sex (no, % male) Race (no, % Caucasian) Donor type (no, %) Living related donor Living unrelated donor Cadaveric donor Biopsy-proven MN in native kidney (no, %) Prior transplants (no, %) Standard triple immunosuppressant therapy (no, %) Induction with Thymoglobulin (no, %)
Nonrecurrent n ¼ 8
Recurrent n ¼ 18
p-value*
41.5 11.3 52.5 10.5 6 (75%) 8 (100%)
41.7 13.6 51.3 11.1 11 (61%) 15 (83%)
0.87 0.80 0.67 0.53
3 (37.5%) 1 (12.5%) 4 (50%) 8 (100%) 0 6 (75%) 6 (75%)
10 (56%) 6 (33%) 2 (11%) 16 (89%) 4 (22%) 15 (83%) 14 (77%)
0.135
1.0 0.28 0.63 1.0
Mean standard deviation unless otherwise indicated. * Comparisons in baseline characteristics were performed by unpaired t-test, two tailed U test (Mann–Whitney) or Wilcoxon signed-rank test for continuous variables and Fisher’s exact test for binary variables.
Comparison of anti-PLA2R Western blotting and ELISA Using the manufacturer’s definition for anti-PLA2R negativity as 3 g/day and he was treated with a second course of RTX 3 years post-Tx, this time with achievement of a CR despite continued presence of the antibodies. Anti-PLA2R eventually resolved 3 years after the second RTX dose. His proteinuria worsened a third time to >3 g/day requiring a third RTX course. His protocol biopsies have shown features of ongoing MN on biopsy including positive PLA2R tissue staining (Figure 5). Positive pre-Tx anti-PLA2R without recurrent MN Two patients [20, 23] had strongly positive pre-Tx antiPLA2R but have not demonstrated evidence of recurrent MN on any protocol biopsies, starting at the 4-month point. However, in both cases, the first post-Tx serum sample was not available for anti-PLA2R testing until 2 and 5 years later, respectively, and both were seronegative. American Journal of Transplantation 2015; 15: 1349–1359
Negative pre-Tx anti-PLA2R with and without recurrent MN All seven patients with recurrent MN and five with no recurrence who were seronegative for anti-PLA2R pre-Tx remained seronegative during the post-Tx course.
Prior history of positive anti-PLA2R, negative pre-Tx with recurrent MN Patient 18 is unique in that this was his second Tx, as his first was lost due to venous thrombosis. Prior to his first Tx, he was anti-PLA2R seropositive, but during the short period between transplants, the antibody disappeared such that he was seronegative at the time of his second Tx. His 4-month protocol biopsy showed recurrent MN with positive PLA2R tissue staining, although his serum remained negative for anti-PLA2R and he has not developed significant proteinuria.
Discussion The primary outcome of this study to determine the predictive value of pre-Tx anti-PLA2R in recurrent MN is that recurrence occurs in a high proportion of patients with positive pre-Tx antibody (83%), but also that the risk of recurrence is as high as 58% in those that are seronegative pre-Tx (Figure 1). In our cohort, the PPV of positive pre-Tx anti-PLA2R for recurrent MN is 83% and the NPV is 42%. Though the number of patients is small, further analysis of follow up data allows us to make 1355
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Figure 4: Patient 5. Recurrence diagnosed at 0.3 months post-Tx. (A) Changes in proteinuria, semi-quantitative Ab levels and IgG staining by immunofluorescence (IF) over time. (B) Weakly-positive PLA2R staining at 0.3 months post-Tx in setting of antibodymediated rejection. (C) Positive PLA2R staining at 18 months postTx.
broader statements that will be of value in the future. Although a positive test for anti-PLA2R pre-Tx predicts early recurrence of MN, in some cases this is a subclinical biopsy finding that may resolve with standard post-Tx immunosuppression. It has been previously established that symptomatic recurrence may lead to graft loss (12,13,21–23). In this study graft loss was an infrequent outcome (3/18 patients) because most cases of recurrence that were detected by protocol biopsy were effectively treated with RTX if they developed proteinuria (11/18 patients). A particularly informative subset of patients was those that were seropositive pre-Tx and became seronegative without additional immunosuppression [14, 18, 20, 23]. In these cases, prospective serial measurements showing decline and disappearance of anti-PLA2R could be used clinically in place of biopsies to monitor for recurrent disease. We propose that patients with primary MN may be separated into two distinct groups—those with PLA2Rassociated MN and those with non-PLA2R-associated MN. We define those with PLA2R-associated MN in this 1356
Figure 5: Patient 8. Recurrence diagnosed at 3 months post-Tx. (A) Changes in proteinuria, semi-quantitative Ab levels and IgG staining by immunofluorescence (IF) over time. (B) Positive PLA2R staining at 16 months post-Tx, after RTX. (C) Positive PLA2R staining 5 years post-Tx, after two courses of RTX.
study as patients with a positive serum anti-PLA2R either pre- or post-Tx and/or positive staining for PLA2R in kidney tissue in the native biopsy or at the time of recurrence. Patients with documented native or recurrent MN on biopsy with consistently negative serum and negative tissue staining are considered to have non-PLA2Rassociated MN. Those patients who were seronegative pre-Tx and had no evidence of recurrence or no tissue available for staining at time of recurrence could potentially fall into either group, only prior information on anti-PLA2R or PLA2R staining of the native kidney biopsy at the time of original diagnosis could have distinguished them. Due to the lack of serum and limited availability of tissue samples from the time when patients were initially diagnosed with MN in their native kidneys, the recurrence rate amongst those with PLA2R-associated and non-PLA2R associated MN cannot be determined in this cohort, although there was close concordance between the native kidney PLA2R positivity and pretransplant anti-PLA2R status in the few cases in which tissue American Journal of Transplantation 2015; 15: 1349–1359
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was available. However, we were able to identify a single subject [22] whose native biopsy stained for PLA2R thus establishing the disease as PLA2R-associated MN. The finding of seronegativity at transplantation suggests a lack of immunologic activity, and is likely the reason why this patient had no evidence of recurrence. Our study includes 18 patients with recurrence: 12 of them with PLA2R-associated MN (66%), four with non-PLA2R associated MN (22%), and two with unknown status (11%). This rate is compatible with the prevalence of antiPLA2R in patients with primary MN. The overall recurrence rate in our cohort (57%) is higher than in previously published series (30–42%) (13,24), but could be related to earlier histologic diagnosis with protocol biopsies, longer follow up of our cohort, and the high number of living related donor transplants in this cohort (25). Our biopsy protocol also provided a unique opportunity to verify whether the IgG1 subclass is dominant and predates IgG4 deposition in glomeruli as suggested by Huang et al in native biopsies (20). Of the seven patients (regardless of PLA2R status) that had biopsies available for IgG subtyping at the first time histologic recurrence was noted [5, 6, 11, 14–17], 6/7 had predominantly IgG4 staining in capillary walls (Table S2). Similarly, IgG4 was the only or dominant subclass in most of the earliest biopsies of subjects with PLA2R-associated MN [5, 6, 9, 11, 14, 15]. A single case of known non-PLA2R-associated MN [16] had only IgG1 staining. Recurrent MN In patients with positive pre-Tx anti-PLA2R, the antibody can disappear within 13 months or less after Tx, which may be due to standard post-Tx immunosuppression, additional RTX treatment, or unknown factors related to antigen exposure in the allograft. Early recurrence that resolved spontaneously could have been missed in some cases prior to the first protocol biopsy. In addition, a few patients were seronegative at time of recurrence and had positive tissue staining for PLA2R [11, 13, 14, 18]. In two of them [11, 14] one might argue that their high pre-Tx antibody levels induced an early recurrence but were no longer detectable in the serum by the time of the first protocol biopsy. Such reasoning cannot apply to patient 13 who recurred after 26 months and whose 4-month protocol biopsy did not have evidence of MN. Nor does it explain why patient 18 developed PLA2R-associated recurrent MN in his second allograft at a time when he was apparently seronegative and thus his kidney was never exposed to detectable antiPLA2R. This finding of negative anti-PLA2R in the serum with positive tissue staining is consistent with prior studies (4) and may reflect very low levels of circulating antibodies that are consumed by the allograft and thus are undetectable by the most sensitive methods available. These low levels may be able to accumulate in the American Journal of Transplantation 2015; 15: 1349–1359
glomeruli over time and not only cause histological changes detectable by immunostaining but can also induce clinical recurrence and graft loss despite apparent seronegativity. These data also suggest that tissue staining for PLA2R may be a more sensitive modality for detecting early recurrence, though this method may be less useful in centers that do not perform protocol biopsies. The persistence or reappearance of anti-PLA2R may herald a more aggressive disease. In six patients [5, 7–10, 15], antiPLA2R levels remained positive or re-appeared years after Tx and were accompanied by symptomatic disease with significant proteinuria. Another insight from this study is that about half of those patients that were seronegative pre-Tx and post-Tx went on to develop symptomatic recurrent MN. As up to 20% of patients with idiopathic MN may have non-PLA2R-associated disease, we would expect that some of the patients in our cohort also have MN associated with one or more distinct, and as yet unidentified, podocyte antigens. However, assuming that at least some of these seronegative cases did indeed have PLA2R-associated MN in immunologic remission at the time of transplantation, this study provided an opportunity to determine if they reactivated when exposed to the new allograft. Notably, none of these cases became seropositive post-Tx, and none of those with available tissue had positive PLA2R immunostaining. These results suggest that post-Tx antiPLA2R testing is of little value in cases that are seronegative pre-Tx unless they are known (from prior serological studies or PLA2R staining of their native kidney) to have PLA2Rassociated MN. Nonrecurrent MN There were two patients that were seropositive for antiPLA2R pre-Tx that became seronegative post-Tx and had no evidence of recurrent MN on protocol biopsy, which suggests that the antibodies either failed to transfer the disease to the allograft or Tx immunosuppression rapidly induced suppression of anti-PLA2R production. In those patients seronegative for anti-PLA2R at the time of Tx, a proportion were likely cases of PLA2R-associated who were immunologically inactive, such as patient 22 who was positive for the PLA2R antigen on native biopsy but seronegative at the time of Tx. While in anti-glomerular basement membrane (GBM) disease, the recurrence rate is significantly reduced if circulating anti-GBM antibody levels have been undetectable for at least 12 months before Tx (26), the optimal duration of seronegativity in PLA2Rassociated MN is unknown. Our findings are consistent with the findings of Debiec et al, who have previously shown that the kinetics of anti-PLA2R post-Tx is quite variable and we found similar patterns of change anti-PLA2R post-Tx (4). Our study has the advantage of using protocol biopsies, allowing us to identify 1357
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subclinical, histologic recurrence of MN, as well as more complete serologic follow up of anti-PLA2R, including both pre- and post-Tx samples. Our study has several limitations. It is a small, retrospective study and so our analysis is limited by the availability of serum and tissue samples. The data is quite heterogeneous and our power to detect significant differences between groups is limited. In addition, we are unable to comment on the overall recurrence rate in PLA2R-associated versus nonPLA2R-associated MN due to limited native kidney biopsy specimens. In conclusion, pre-Tx anti-PLA2R testing may have clinical utility either in those with known PLA2R-associated disease or those who are seropositive at the time of transplant. Now that the assays are more widely available in the clinical setting, anti-PLA2R should be checked in all patients with primary MN being considered for transplantation and the native kidney biopsy should be stained for PLA2R in order to classify them as having PLA2R- or nonPLA2R-associated MN prior to transplantation. The presence of anti-PLA2R at the time of Tx very often leads to early recurrence of MN. Induction therapy and standard maintenance immunosuppression in some cases are sufficient to induce immunologic remission, but do not preclude the possibility of recurrence of PLA2R-associated MN. In those cases with persistence or reappearance of the antibody post-Tx the disease appears more resistant. The response to RTX is excellent and should be considered to prevent allograft loss. Our study raises two practical issues. One is whether to postpone Tx until the circulating anti-PLA2R disappears and for how long to wait with an undetectable level before Tx? Second, considering the high recurrence rate documented in patients seropositive for anti-PLA2R at the time of transplantation, should RTX be given preventively to all patients with either pre- or post-Tx anti-PLA2R seropositivity? Future prospective studies are needed to establish the answers to these questions.
Acknowledgments We would like to thank Dr. Joel Henderson from the Department of Pathology and Laboratory Medicine at Boston University Medical Center for guidance and advice with the tissue staining. This work is supported by R01 DK090029 (DJS, LHB), R01 DK 097053 (LHB) from NIH/NIDDK and NEPTUNE, Nephrotic Syndrome Study Network, U-54-DK-083912 (RA).
Disclosure The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. LHB and DJS are co-inventors on the US patent, Diagnostics for Membranous Nephropathy. 1358
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Supporting Information Additional Supporting Information may be found in the online version of this article. Supplementary Material and Methods Figure S1: Correlation between Western blot (semiquantitative scale) and ELISA. ELISA values were logarithmically transformed due to non-normal distribution. Table S1: HLA typing in recipients and donors. Table S2: PLA2R antigen staining on native biopsies and IgG subtyping in posttransplant biopsies in patients with MN recurrence. Table S3: Anti-PLA2R results by Western blotting (semiquantitative compared to well-characterized positive control) and by ELISA (RU/mL; negative
